Design of a herpes simplex virus type 2 long oligonucleotide-based microarray: global analysis of HSV-2 transcript abundance during productive infection.

The design and construction of a long (75-mer) oligonucleotide-based DNA microarray for herpes simplex virus type 2 transcripts is described. This array is utilized to generate an analysis of HSV-2 transcript abundance as a function of conditions of infection of human cells, and global patterns of HSV-2 transcript abundance are compared with those for HSV-1. General similarities in patterns along with notable differences in specific details are noted. These results reveal a marked conservation in the program of gene activity between phenotypically diverged strains.

Inhibition of muscarinic cholinergic receptors by disulfide reducing agents and arsenicals. Differential effect on locust and rat.

Muscarinic receptors are altered by sulfhydryl reagents. Arsenic compounds, which have been used as insecticides, exert their toxic effects by combining with sulfhydryl groups. We compared the action of arsenicals and other sulfhydryl reagents on the muscarinic receptor from invertebrate and vertebrate species (locust and rat). Disulfide-reducing reagents dithiothreitol (DTT) and British Anti-Lewisite (BAL), but not arsenicals, inhibited [3H]quinuclidinyl benzilate ([3H]QNB) binding. However, after disulfide reduction, arsenicals caused a further inhibition of muscarinic binding. The effect of DTT + arsenicals was largely irreversible. The locust receptors were more sensitive to the action of both disulfide reagents either in the absence or presence of arsenicals than the rat receptors. The sulfhydryl reagent p-chloromercuric benzoate (PCMB) was more effective at inhibiting the locust receptors than the rat receptors, but addition of arsenicals did not cause further inhibition in either the locust or rat receptors. In locust, DTT + cacodylate and DTT + arsenite caused a reduction in the number of sites without modifying the affinity of [3H]QNB binding. In rat, DTT + arsenite caused a decrease in the affinity, while DTT + cacodylate caused a decrease in the affinity of [3H]QNB binding and its number of sites. Competition experiments after DTT + cacodylate showed that the IC50 and the Hill coefficient (nH) remained unchanged in the locust. In the rat, the IC50 for atropine was increased without alteration in the nH, and both parameters were increased for carbachol. These results are explained assuming that the binding site of the locust receptor has a disulfide group similar to that of the mammalian receptor, but that the hydrophobic interactions within the binding site are weaker in the locust receptor. The higher sensitivity of the insect receptor to sulfhydryl reagents could be of interest for developing methods of pest control.

The heterogeneity and nucleotide modulation of cholinergic muscarinic receptors are restored by poly (ethylene) glycol-6000 precipitation of solubilized atrial receptors.

The membrane bound muscarinic receptor exhibits heterogeneity of binding sites for agonists that are modulated by guanine nucleotides. These properties are generally lost upon receptor solubilization by several detergents, but may be recovered after rapid detergent removal by poly (ethylene) glycol-6000 precipitation of the digitonin plus cholate solubilized atrial muscarinic receptor. The results are discussed in terms of muscarinic receptor interactions in the intact membrane.

Muscarinic receptor sites in human foetal brain.

The specific binding of the muscarinic cholinergic ligand N-methylscopolamine to human foetal brain has been measured. A level of binding of 64 pmol/g protein was found with a dissociation constant, K(d) of 0.27 nM. Values of 0.17 nM min(?1) and 0.048 min(?1) for the association rate constant, K(on), and the dissociation rate constant K(off) respectively, were obtained. The pharmacological properties of the binding site were found to be very similar to those reported for muscarinic receptors from adult mammalian brain except that the binding of pirenzepine and the nicotinic antagonists d-tubocurarine and decamethonium shows differences from that seen in adult brain.

Cholinergic binding sites with muscarinic properties on membranes from the supraoesophageal ganglion of the locust (Schistocerca gregaria).

A crude membrane preparation from the supraoesophageal ganglion of the locust (Schistocerca gregaria) shows specific binding of muscarinic cholinergic ligands. Analysis of the kinetics of binding reveals the presence of at least two binding sites with dissociation constants, K(d) of 0.76 and 37.7 nM. The pharmacological profile of the higher affinity site is different from that seen for muscarinic receptor sites in mammalian brain. The binding sites reported here are quite distinct from nicotinic-like receptor sites in the same tissue and lend further support to suggestions that there are at least two types of acetylcholine receptors in insects.

Cellular mapping of m2 muscarinic receptors in rat olfactory bulb using an antiserum raised against a cytoplasmic loop peptide.

An antiserum that recognizes a sequence from the putative third cytoplasmic loop of the m2 subtype of muscarinic receptors (mAchR) has been raised and used to map the cellular distribution of this subtype in rat olfactory bulb. The antiserum was obtained by injecting BALB/C mice with a BSA-conjugated synthetic peptide whose sequence corresponded to amino acids 240-259 of the porcine cardiac m2 mAChR gene. Antibodies recognized the synthetic peptide in ELISA screening and labelled a single band corresponding to the peak of [3H]PrBCM-labelled heart mAchRs in immunoblots. Immunostaining of olfactory bulb, a region of the brain enriched in this muscarinic receptor subtype, showed that the antibodies labelled cell bodies and multiple dendritic processes. Broad fluorescent labelling throughout cell bodies was consistent with binding to the cytoplasmic face of the surface membrane, in support of the predicted cytoplasmic loop structure. m2-Positive cells throughout the bulb were sparsely distributed in different layers representing small subpopulations of the cells in each region: glomeruli, 6%; external plexiform layer, 16%; inner plexiform and granule cell layer, 3%. The results show that antibodies against specific sequences of different muscarinic receptor subtypes can be used to localize subtypes in situ, that the m2 subtype within the rat olfactory bulb is broadly distributed, and that the m2 subtype can occur postsynaptically in this central nervous system (CNS) region. The mapping of m2-positive cells in olfactory bulb may be of particular interest because loss of this subtype and degeneration of the olfactory system have been observed in Alzheimer's disease.

Localization of hippocampal muscarinic receptors after kainic acid lesion of CA3 and fimbria-fornix transection.

Bilateral intraventricular injections of 0.5 microgram of kainic acid were used to selectively destroy CA3 hippocampal pyramidal neurons, in an effort to clarify the possible localization of muscarinic cholinergic receptors in the rat hippocampal formation. Thirty days after treatment, there was 43% decrease in the total number of [3H]L-QNB binding sites per hippocampus, with no change in affinity. Histological examination confirmed the selective loss of pyramidal neurons in subareas CA3a-b while other regions of the hippocampal formation were spared. The unilateral transection of the fimbria-fornix, done 14 days after kainic acid, produced a further reduction in binding in relation to control hippocampi (-57%). The results demonstrate that in the pyramidal cells of CA3 there is a high concentration of postsynaptic muscarinic receptors. However, the slight further decrease, found after fimbria-fornix transection, suggests the possible existence of a small population of presynaptic receptors that, hitherto, had only been demonstrated indirectly by physiological methods.

Inhibition by local anesthetics, phentolamine and propranolol of [3H]quinuclydinyl benzylate binding to central muscarinic receptors.

[3H]Quinuclydinyl benzylate ([3H]QNB) binding was carried out on crude synaptosomal membranes isolated from cat cerebral cortex. The specific binding showed a single type of site with KD 0.25 nM, Hill number 0.89 and Bmax 114 pmol/g protein. The local anesthetics procaine, tetracaine and dibucaine, and the adrenergic antagonists phentolamine and propranolol, in concentrations between 1 nM and 500 microM, inhibited [3H]QNB binding with Ki varying between 9 microM for procaine and 80 microM for propranolol. The Hill coefficients obtained from logit/log plots suggested that there was no cooperativity between the binding sites for local anesthetics. At various concentrations the inhibition by procaine and propranolol may appear as competitive or non-competitive. The Hill numbers obtained from the saturation curves suggest that there was no cooperativity between anesthetics and [3H]QNB binding sites. Neither 1 mM Ca2+ nor Mg2+ affected [3H]QNB binding or the action of the drugs. The effect of local anesthetics and adrenergic antagonists was reversible and these drugs did not protect the muscarinic receptor from the deleterious effect of Triton X-100 as was the case with muscarinic agents. Our findings suggest that local anesthetics inhibit [3H]QNB binding to the muscarinic receptor by acting at some accessory site but not on the true receptor site. The possible mechanism of action and local anesthetics on synaptic transmission is discussed.

Action of detergents on 3H-dihydroergokriptine binding and localization of alpha-adrenoceptors in synaptosomal membranes.

3H-dihydroergokriptine (3H-DHE) binding was carried out in synaptosomal membranes from basal ganglia of the cat. A single type of binding site with Kd 3.7 nM, Hill number = 0.95 and Bmax = 1000 pmol/g protein was found. 3H-DHE bound to alpha-adrenoceptors and not to serotonin or dopamine receptors. At very low concentrations, some detergents enhanced binding, but at higher concentrations of those used (Triton X-100, Nonidet P-40, deoxycholate and digitonin), inhibited the binding of 3H-DHE. After binding to the membrane protein, the 3H-DHE-receptor complex was stable to the action of Triton X-100. At concentrations of Triton X-100. At concentrations of Triton X-100 (0.1--0.2%). in which only the presynaptic membrane disintegrated, the 3H-HDE specific radioactivity was reduced. With a more drastic treatment that disintegrated the postsynaptic membrane, 3H-DHE binding was further reduced. These results suggest that alpha-adrenergic receptors may be localized at both the pre- and postsynaptic membranes of central synapses.

Protection by atropine of the inhibition caused by Triton X-100 on central muscarinic receptors.

Synaptosomal membranes from cat cerebral cortex were labelled with [3H]-quinuclidinyl benzylate ([3H]QNB). There was shown to be a single type of binding site with Kd = 0.34 nM, Bmax = 2.2 nmol/g protein and Hill No. = 1.01. Triton X-100 at 5 x 10(-4)% inhibited the specific binding of [3H]QNB and the inhibition was almost complete at 10(-2)%. Treatment with 2.5 x 10(-6) M atropine, followed by centrifugation washings protected the receptor site from the inhibitory action of the detergent. The protection afforded by other cholinergic drugs was less effective. The use of this technique has confirmed the results of our previous work on the possible pre- and postsynaptic location of central muscarinic receptors. These findings open the possibility for protection of other detergent-sensitive receptors and for their isolation and purification as well-defined macromolecules.

Pre- and postsynaptic localization of central muscarinic receptors.

3H-Quinuclidinyl benzylate and dimethyl 14C-d-tubocurarine were used to localize muscarinic and nicotinic receptors in synaptosomal membranes of the rat cerebral cortex. The results obtained, after the action of different concentrations of Triton X-100, suggest that, while nicotinic receptors are postsynaptic, the muscarinic ones are both pre- and postsynaptic.

Changes in rat hippocampal benzodiazepine receptors and lack of changes in muscarinic receptors after fimbria-fornix lesions.

The binding of [3H] L-quinuclidinylbenzilate to muscarinic receptors and [3H] flunitrazepam to benzodiazepine receptors were studied in the rat hippocampus after lesion of the fimbria-fornix. While the muscarinic receptors showed no change, the benzodiazepine receptors did change considerably at various time intervals. Two days after the lesion the specific [3H] flunitrazepam binding decreased 38%, while at 5 days it increased 65%. After 14 days of the lesion it still was significantly above normal. These changes are due to a variation in the number of sites, and not in affinity. Possible interpretations of these results are discussed.

Receptor binding properties of di (1,N6-ethenoadenosine) 5', 5'''-P1, P4-tetraphosphate and its modulatory effect on extracellular glutamate levels in rat striatum.

Our aim was to investigate the neuromodulatory role of diadenosine tetraphosphate (Ap(4)A). Ap(4)A-binding sites were detected in striatum and hippocampus membranes using [(35)S]-ADP beta S as radioligand and Ap(4)A and epsilon-(Ap(4)A), di-ethenoadenosine tetraphosphate, as displacers. Effects of epsilon-(Ap(4)A) on extracellular glutamate levels were studied using intracerebral perfusion. Both areas contain high-affinity binding sites for [(35)S]-ADP beta S with K(d) values in the low nM range. [(35)S]-ADP beta S binding was displaced by Ap(4)A and epsilon-(Ap(4)A). At 1 and 10 microM doses, epsilon-(Ap(4)A) markedly decreased glutamate levels in the striatum. The possibility of Ap(4)A acting as an endogenous modulator of excitatory neurotransmission is discussed.

Dimethyl sulfoxide blocks herpes simplex virus-1 productive infection in vitro acting at different stages with positive cooperativity. Application of micro-array analysis.

BACKGROUND: Dimethyl sulfoxide (DMSO) is frequently used at a concentration of up to 95% in the formulation of antiherpetic agents because of its properties as a skin penetration enhancer. Here, we have analyzed the effect of DMSO on several parameters of Herpes Simplex Virus replication. METHODS: Productive infection levels of HSV-1 were determined by plaque assay or by reporter gene activity, and its DNA replication was estimated by PCR. Transcript levels were evaluated with HSV-specific DNA micro-arrays. RESULTS: DMSO blocks productive infection in vitro in different cell types with a 50% inhibitory concentration (IC50) from 0.7 to 2% depending upon the multiplicity of infection. The concentration dependence exhibits a Hill coefficient greater than 1, indicating that DMSO blocks productive infection by acting at multiple different points (mechanisms of action) with positive cooperativity. Consistently, we identified at least three distinct temporal target mechanisms for inhibition of virus growth by DMSO. At late stages of infection, DMSO reduces virion infectivity, and markedly inhibits viral DNA replication. A third mode of action was revealed using an oligonucleotide-based DNA microarray system for HSV. These experiments showed that DMSO reduced the transcript levels of many HSV-1 genes; including several genes coding for proteins involved in forming and assembling the virion. Also, DMSO markedly inhibited some but not all early transcripts indicating a previously unknown mode for inhibiting the early phase of HSV transcription-replication cycle. CONCLUSION: These observations suggest that DMSO itself may have a role in the anti-herpetic activity of formulations utilizing it as a dispersant.

Quantitative comparison of the HSV-1 and HSV-2 transcriptomes using DNA microarray analysis.

The genomes of human herpes virus type-1 and type-2 share a high degree of sequence identity; yet, they exhibit important differences in pathology in their natural human host as well as in animal host and cell cultures. Here, we report the comparative analysis of the time and relative abundance profiles of the transcription of each virus type (their transcriptomes) using parallel infections and microarray analysis using HSV-1 probes which hybridize with high efficiency to orthologous HSV-2 transcripts. We have confirmed that orthologous transcripts belong to the same kinetic class; however, the temporal pattern of accumulation of 4 transcripts (U(L)4, U(L)29, U(L)30, and U(L)31) differs in infections between the two virus types. Interestingly, the protein products of these transcripts are all involved in nuclear organization and viral DNA localization. We discuss the relevance of these findings and whether they may have potential roles in the pathological differences of HSV-1 and HSV-2.

Atrial muscarinic receptors: effect of Triton X-100 on guanine nucleotide and ammonium ion modulation.

Membranes isolated from bovine atria were labeled with [3H]quinuclidinyl benzylate (3H-QNB), in control conditions and after 0.02% Triton X-100. This treatment inactivated about 20% of muscarinic receptor sites without loss of protein. The remaining 80% sites showed no changes in affinity, as determined by equilibrium or kinetic binding. Competition experiments with carbachol showed no differences in IC50 and Hill number between the control and detergent-membranes, suggesting that the different populations of agonist binding sites are inactivated in equal proportions by the detergent. In binding experiments, done in the presence of carbachol and guanine nucleotides, the detergent treated membranes were slightly more sensitive to the enhancing action of the nucleotide. The inhibition caused by ammonium ions was also more marked in the Triton X-100 treated membranes. The decay of binding with thermal inactivation was faster in the detergent treated membranes and this effect was enhanced in the presence of ammonium ions. These results may be interpreted as an indication that the receptors, remaining after the mild Triton X-100 treatment, are equally sensitive to the inactivation. We suggest that, while maintaining the heterogeneity of sites, the detergent produces a perturbation that could affect the molecular interactions between the receptor and other components of the membrane.

Reconstitution of solubilized atrial cholinergic muscarinic receptors in liposomes.

The reconstitution of solubilized bovine atrial cholinergic muscarinic receptor into liposomes made of exogenous lipids has been achieved by polyethyleneglycol precipitation. Of the different lipid mixtures used, soybean lecithins were shown to be the best on the basis of receptor recovery. The receptor reconstituted into soybean lecithins liposomes exhibited ligand binding properties very similar to those of the native receptor. The dissociation constant of [3H]-N-methyl-scopolamine ([3H]NMS) was 0.46 and 0.30 nM as determined by equilibrium and kinetics experiments respectively. The potency of a range of muscarinic ligands in displacing [3H]NMS binding was atropine greater than methyl-atropine greater than scopolamine greater than pirenzepine oxotremorine greater than gallamine greater than carbamylcholine greater than pilocarpine bethanechol. The Hill slopes of the displacement curves were near 1 for the antagonists and smaller than 1 for the agonists and for gallamine. The agonist binding may be modulated by guanine nucleotides. These results indicate that soybean lecithins fulfill the lipid requirements for the reconstitution of the atrial muscarinic receptor.

Pertussis vaccine reduces agonist binding to the rat heart muscarinic receptor and its guanine nucleotide modulation.

The injection of Bordetella pertussis, inactivated by merthiolate, causes a 2-fold increase in the IC50 of carbamylcholine (carbachol) in displacing [3H];-L(-) quinuclidinyl benzilate binding ([3H]QNB) to the receptor. In control animals, 50 microM Gpp(NH)p causes a 6-fold decrease in the affinity of carbachol binding, whereas after vaccination the reduction is only 1.6-fold. After pertussis treatment there is no alteration in the affinity and number of [3H]QNB binding sites of to the muscarinic receptor.

Sulphydryl groups and iodo-[3H]acetic acid labeling in proteolipids from Torpedo electroplax.

Several fractions of proteolipids from Torpedo electroplax were separated by DEAE-cellulose chromatography in organic solvents, and the sulphydryl groups were determined by a spectrophotometric method. On the same fractions the covalent labeling with iodo-[3H]acetic acid to sulphydryl groups was studied. In total proteolipids there were 30.3 nmol/mg protein of sulphydryl groups of which 20.6 nmoles were in the form of disulfide bonds and 10.9 nmol as free--SH groups. The highest content of sulphydryl groups (36.7 nmol/mg protein) was found in fraction II; while fraction I, that binds the cholinergic ligands, has a lower content (23.7 nmol/mg protein). The 42 Kdaltons polypeptide, which is the major band in Fraction II, has the strongest labeling with iodo-[3H]acetic acid, while the 39 Kdaltons cholinergic polypeptide shows a lower labeling. The importance of proteolipids as channel-forming macromolecules is discussed in connection with the possible significance of the 42 Kdaltons polypeptide.

The polysulfonated compound suramin blocks adsorption and lateral difusion of herpes simplex virus type-1 in vero cells.

Several polysulfonate compounds have been shown to have the potential to inhibit the replication of herpesviruses by blocking binding and penetration of the host cell. We analyzed the actions of the polysulfonate compound suramin on the replication of herpes simplex virus type 1 (HSV-1) and compared them with the actions of heparin. We used the expression of a reporter gene (beta-galactosidase) recombined into the latency-associated transcript region of the 17syn+ strain of HSV-1 to quickly evaluate productive cycle activity and have shown that it can be directly correlated with virus replication under the conditions used. We find that suramin, like heparin, blocks the binding of HSV-1 to the cell membrane. Also, suramin efficiently blocks the cell-to-cell spread of the virus; this effect has not been previously reported. Our control experiments demonstrate that heparin also has some effect on intercellular spread of HSV-1 but to a significantly lesser degree than does suramin. We suggest that suramin and related polysulfonate compounds have potential for developing of antiherpes treatments.